In Drosophila sex is determined by the ratio of the number of X chromosomes to the number of sets of autosomes. Cells with one X chromosome and two sets of automsomes are males and cells with 2 X chromosomes and two sets of autosomes are females. Since males have the same requirements for X-linked gene products as females, they respond by hyperactivating the X chromosome at the transcriptional level to compensate for the difference in gene dosage. This project is aimed at two questions: how do male cells double the transcription rate of virtually every gene on the X chromosome, and how do cells count the number of X chromosomes. The first question will be addressed by using the gene for 6-phosphogluconate dehydrogenase (Pgd) as a model gene for the X chromosome. In vitro manipulation of the gene and its promoter, followed by reintegration into the Drosophila genome, will be used to find the cis-acting regulatory site of the Pgd gene that is responsible for the dosage compensation response. Dosage compensation is an important topic in the study of gene regulation since it changes the expression levels of a large battery of genes by exactly two-fold. This subtle regulation may be controlled by a novel mechanism, or by novel application of conventional mechanisms. Understanding dosage compensation will be an important step in understanding the coordinated regulation of a large group of genes, which is also a significant facet of development and neoplasia. The question of how the X chromosomes are counted will be attacked by cloning the Tp1 gene either by a chromosome walk or transposon tagging, and then studying the expression of the gene. Tpl is a locus on chromosome 3 which is extremely dosage sensitive. Drosophila requires exaclty 2 doses of Tpl for survival. Individuals bearing 1 or 3 doses die as embryos. It has been suggested that Tpl is involved in counting the X chromosomes for two reasons. One would expect a gene that titrates the X chromosomes against itself to be dosage sensitive, and in addition, Tpl appears to interact with the X in aneuploids. If this hypothesis turns out be incorrect, then Tpl must perform a vital function in embryogenesis or cellular metabolism. In either case the study of Tpl has for-reaching implications for sex determination, the consequences of aneuploidy, and embryogenesis. Drosophila is one of very few organisms in which such a gene can be studied since it has a dominant lethal phenotype.